Murine pregnancy-specific glycoprotein 23 induces the proangiogenic factors transforming-growth factor beta 1 and vascular endothelial growth factor a in cell types involved in vascular remodeling in pregnancy

Haemochorial placentation is a unique physiological process in which the fetal trophoblast cells remodel the maternal decidual spiral arteries to establish the fetoplacental blood supply. Pregnancy-specific glycoproteins (PSGs) are members of the carcinoembryonic antigen family. PSGs are produced by the placenta of rodents and primates and are secreted into the bloodstream. PSG23 is one of 17 members of the murine PSG family (designated PSG16 to PSG32). Previous studies determined that PSGs have immunoregulatory functions due to their ability to modulate macrophage cytokine secretion. Here we show that recombinant PSG23 induces transforming growth factor (TGF) beta1, TGFB1, and vascular endothelial growth factor A (VEGFA) in primary murine macrophages and the macrophage cell line RAW 264.7 cells. In addition, we identified new cell types that responded to PSG23 treatment. Dendritic cells, endothelial cells, and trophoblasts, which are involved in maternal vasculature remodeling during pregnancy, secreted TGFB1 and VEGFA in response to PSG23. PSG23 showed cross-reactivity with human cells, including human monocytes and the trophoblast cell line, HTR-8/SVneo cells. We analyzed the binding of PSG23 to the tetraspanin CD9, the receptor for PSG17, and found that CD9 is not essential for PSG23 binding and activity in macrophages. Overall these studies show that PSGs can modulate the secretion of important proangiogenic factors, TGFB1 and VEGFA, by different cell types involved in the development of the placenta.     

Pregnancy-specific glycoprotein 1 induces endothelial tubulogenesis through interaction with cell surface proteoglycans

Pregnancy-specific β1 glycoproteins (PSGs) are the most abundant fetal proteins in the maternal bloodstream in late pregnancy. They are secreted by the syncytiotrophoblast and are detected around day 14 postfertilization. There are 11 human PSG genes, which encode a family of proteins exhibiting significant conservation at the amino acid level. We and others have proposed that PSGs have an immune modulatory function. In addition, we recently postulated that they are proangiogenic due to their ability to induce the secretion of VEGF-A and the formation of tubes by endothelial cells. The cellular receptor(s) for human PSGs remain unknown. Therefore, we conducted these studies to identify the receptor for PSG1, the highest expressed member of the family. We show that removal of cell surface glycosaminoglycans (GAGs) by enzymatic or chemical treatment of cells or competition with heparin completely inhibited binding of PSG1. In addition, PSG1 did not bind to cells lacking heparan or chondroitin sulfate on their surface, and binding was restored upon transfection with all four syndecans and glypican-1. Importantly, the presence of GAGs on the surface of endothelial cells was required for the ability of PSG1 to induce tube formation. This finding indicates that the PSG1-GAG interaction mediates at least some of the PSG1 proposed functions.     

The balance of proangiogenic and antiangiogenic VEGFA isoforms regulate follicle development

Vascular endothelial growth factor A (VEGFA) has been extensively studied because of its role in follicular development and is a principal angiogenic factor essential for angiogenesis. Since vascularization of the theca layer increases as follicles progress in size through preantral and antral stages, VEGFA might influence follicle growth via the regulation of angiogenesis. However, VEGFA might also influence follicular development through nonangiogenic mechanisms, since its expression has been localized in nonvascular follicles and cells. Alternative mRNA splicing of eight exons from the VEGFA gene results in the formation of various VEGFA isoforms. Each isoform has unique properties and is identified by the number of amino acids within the mature protein. Proangiogenic isoforms (VEGFA_XXX) are encoded by exon 8a, whereas a sister set of isoforms (VEGFA_XXXB) with antiangiogenic properties is encoded by exon 8b. The antiangiogenic VEGFA_XXXB isoforms comprise the majority of VEGFA expressed in most tissues, whereas expression of the proangiogenic VEGFA isoforms is upregulated in tissues undergoing active angiogenesis. Although proangiogenic and antiangiogenic isoforms can now be distinguished from one another, many studies evaluating VEGFA in ovarian and follicular development up to now have not differentiated proangiogenic VEGFA from antiangiogenic VEGFA. Experiments from our laboratory indicate that proangiogenic VEGFA promotes follicle recruitment and early follicular development and antiangiogenic VEGFA inhibits these processes. The balance of proangiogenic versus antiangiognic VEGFA isoforms is thus of importance during follicle development. Further studies are warranted to elucidate the way that this balance regulates follicular formation and progression.     

Proangiogenic function of CD40 ligand-CD40 interactions

Angiogenesis is a characteristic component of cell-mediated immune inflammation. However, little is known of the immunologic mediators of angiogenesis factor production. Interactions between CD40 ligand (CD40L) and CD40 have been shown to have pluripotent functions in inflammation, including the production of cytokines, chemokines, as well as the angiogenesis factor, vascular endothelial growth factor (VEGF), by endothelial cells. In this study we found that treatment of cultured human endothelial cells with an anti-CD40 Ab (to ligate CD40) resulted in the expression of several other angiogenesis factors, including fibroblast growth factor-2 and the receptors Flt-1 and Flt-4. To determine the proangiogenic effect of CD40L in vivo, human skin was allowed to engraft on SCID mice for 6 wk. These healed human skins express CD40 on resident endothelial cells and monocyte/macrophages, but not on CD20-expressing B cells. Skins were injected with saline, untransfected murine fibroblasts, or murine fibroblasts stably transfected with human CD40L. We found that the injection of CD40L-expressing cells, but not control cells, resulted in the in vivo expression of several angiogenesis factors (including VEGF and fibroblast growth factor) and a marked angiogenesis reaction. Mice treated with anti-VEGF failed to elicit an angiogenesis reaction in response to injection of CD40L-expressing cells, suggesting that the proangiogenic effect of CD40L in vivo is VEGF dependent. These observations imply that ligation of CD40 at a peripheral inflammatory site is of pathophysiological importance as a mediator of both angiogenesis and inflammation.     

PSG9 promotes angiogenesis by stimulating VEGFA production and is associated with poor prognosis in hepatocellular carcinoma

Hepatocellular carcinoma(HCC) is a common malignant solid tumor characterized by rich vascularization. Pregnancy-specific glycoprotein 9(PSG9) is a member of the carcinoembryonic antigen(CEA)/PSG family and is produced at high levels during pregnancy. We previously identified PSG9 as an HCC-related protein. However, the expression of PSG9 and its regulation during HCC carcinogenesis remain poorly explored. In the present study, we first found that the levels of PSG9 protein were significantly increased in the plasma of HCC patients. PSG9 overexpression also increased the proliferation ability of an HCC cell line. High expression of PSG9 was associated with angiogenesis by accelerating VEGFA expression. In addition, Cox's proportional hazards model analysis revealed that the plasma level of PSG9 was an independent prognostic factor for overall survival. We propose that PSG9 is a novel indicator of prognosis in patients with HCC and could serve as a novel therapeutic target for HCC. Furthermore,our results indicate that PSG9 protein may facilitate the development of HCC by fostering angiogenesis via promoting VEGFA production in cancer cells.     

Osteopontin (Eta-1) and fibroblast growth factor-2 cross-talk in angiogenesis

The cytokine/extracellular matrix protein osteopontin (OPN/Eta-1) is an important component of cellular immunity and inflammation. It also acts as a survival, cell-adhesive, and chemotactic factor for endothelial cells. Here, subtractive suppression hybridization showed that serum-deprived murine aortic endothelial (MAE) cells transfected with the angiogenic fibroblast growth factor-2 (FGF2) overexpress OPN compared with parental cells. This was confirmed by Northern blotting and Western blot analysis of the conditioned media in different clones of endothelial cells overexpressing FGF2 and in endothelial cells treated with the recombinant growth factor. In vivo, FGF2 caused OPN expression in newly formed endothelium of the chick embryo chorioallantoic membrane (CAM) and of murine s.c. Matrigel plug implants. Recombinant OPN (rOPN), the fusion protein GST-OPN, and the deletion mutant GST-DeltaRGD-OPN were angiogenic in the CAM assay. Angiogenesis was also triggered by OPN-transfected MAE cells grafted onto the CAM. OPN-driven neovascularization was independent from endothelial alpha(v)beta(3) integrin engagement and was always paralleled by the appearance of a massive mononuclear cell infiltrate. Accordingly, rOPN, GST-OPN, GST-DeltaRGD-OPN, and the conditioned medium of OPN-overexpressing MAE cells were chemotactic for isolated human monocytes. Also, rOPN triggered a proangiogenic phenotype in human monocytes by inducing the expression of the angiogenic cytokines TNF-alpha and IL-8. OPN-mediated recruitment of proangiogenic monocytes may represent a mechanism of amplification of FGF2-induced neovascularization during inflammation, wound healing, and tumor growth.     

MicroRNA-22 regulates hypoxia signaling in colon cancer cells

MicroRNAs (MiRNAs) are short, non-coding RNA that regulate a variety of cellular functions by suppressing target protein expression. We hypothesized that a set of microRNA regulate tumor responses to hypoxia by inhibiting components of the hypoxia signaling pathway. We found that miR-22 expression in human colon cancer is lower than in normal colon tissue. We also found that miR-22 controls hypoxia inducible factor 1α (HIF-1α) expression in the HCT116 colon cancer cell line. Over-expression of miR-22 inhibits HIF-1α expression, repressing vascular endothelial growth factor (VEGF) production during hypoxia. Conversely, knockdown of endogenous miR-22 enhances hypoxia induced expression of HIF-1α and VEGF. The conditioned media from cells over-expressing miR-22 contain less VEGF protein than control cells, and also induce less endothelial cell growth and invasion, suggesting miR-22 in adjacent cells influences endothelial cell function. Taken together, our data suggest that miR-22 might have an anti-angiogenic effect in colon cancer.     

Vascular protection by angiotensin receptor antagonism involves differential VEGF expression in both hemispheres after experimental stroke

We identified that the angiotensin receptor antagonist, candesartan, has profound neurovascular protective properties when administered after ischemic stroke and was associated with a proangiogenic state at least partly explained by vascular endothelial growth factor A (VEGFA). However, the spatial distribution of vascular endothelial growth factor (VEGF) isoforms and their receptors remained unknown. Protein analysis identified a significant increase in vascular endothelial grow factor B (VEGFB) in the cerebrospinal fluid (CSF) and the ischemic hemispheres (with increased VEGF receptor 1 activation) of treated animals (p<0.05) which was co-occurring with an increase in protein kinase B (Akt) phosphorylation (p<0.05). An increase in VEGFA protein in the contralesional hemisphere corresponded to a significant increase in vascular density at seven days (p<0.01) after stroke onset. Vascular restoration by candesartan after stroke maybe related to differential regional upregulation of VEGFB and VEGFA, promoting a "prosurvival state" in the ischemic hemisphere and angiogenesis in the contralesional side, respectively. These vascular changes in both hemispheres after effective treatment are likely to contribute to enhanced recovery after stroke.     

Human B-lymphocytes express alpha2-6-sialylated 6-sulfo-N-acetyllactosamine serving as a preferred ligand for CD22/Siglec-2

CD22/Siglec-2, an important inhibitory co-receptor on B-lymphocytes, is known to recognize alpha2-6-sialylated glycan as a specific ligand. Here we propose that the alpha2-6-sialylated and 6-GlcNAc-sulfated determinant serves as a preferred ligand for CD22 because the binding of a human B-cell line to CD22 was almost completely abrogated after incubating the cells with NaClO3, an inhibitor of cellular sulfate metabolism, and was also significantly inhibited by a newly generated monoclonal antibody specific to the alpha2-6-sialylated 6-sulfo-N-acetyllactosamine (LacNAc) determinant (KN343, murine IgM). The alpha2-6-sialylated 6-sulfo-LacNAc determinant defined by the antibody was significantly expressed on a majority of normal human peripheral B-lymphocytes as well as follicular B-lymphocytes in peripheral lymph nodes. The determinant was also expressed in endothelial cells of high endothelial venules of secondary lymphoid tissues, including lymph nodes, tonsils, and intestine-associated lymphoid tissues, more strongly than on B-lymphocytes, suggesting a role for CD22 in B-cell interaction with blood vessels and trafficking. These results indicate that the alpha2-6-sialylated 6-sulfo-LacNAc determinant serves as an endogenous ligand for human CD22 and suggest the possibility that 6-GlcNAc sulfation as well as alpha2-6-sialylation may regulate CD22/Siglec-2 functions in humans.     

Nerve growth factor translates stress response and subsequent murine abortion via adhesion molecule-dependent pathways

Spontaneous abortion is a frequent threat affecting 10%-25% of human pregnancies. Psychosocial stress has been suggested to be attributable for pregnancy losses by challenging the equilibrium of systems mandatory for pregnancy maintenance, including the nervous, endocrine, and immune system. Strong evidence indicates that stress-triggered abortion is mediated by adhesion molecules, i.e., intercellular adhesion molecule 1 (ICAM1) and leukocyte function associated molecule 1, now being referred to as integrin alpha L (ITGAL), which facilitate recruitment of inflammatory cells to the feto-maternal interface. The neurotrophin beta-nerve growth factor (NGFB), which has been shown to be upregulated in response to stress in multiple experimental settings including in the uterine lining (decidua) during pregnancy, increases ICAM1 expression on endothelial cells. Here, we investigated whether and how NGFB neutralization has a preventive effect on stress-triggered abortion in the murine CBA/J x DBA/2J model. We provide experimental evidence that stress exposure upregulates the frequency of abortion and the expression of uterine NGFB. Further, adhesion molecules ICAM1 and selectin platelet (SELP, formerly P-Selectin) and their ligands ITGAL and SELP ligand (SELPL, formerly P selectin glycoprotein ligand 1) respectively increase in murine deciduas in response to stress. Subsequently, decidual cytokines are biased toward a proinflammatory and abortogenic cytokine profile. Additionally, a decrease of pregnancy protective CD8alpha(+) decidual cells is present. Strikingly, all such uterine stress responses are abrogated by NGFB neutralization. Hence, NGFB acts as a proximal mediator in the hierarchical network of immune rejection by mediating an abortogenic environment comprised of classical signs of neurogenic inflammation.     

Imatinib mesylate (STI-571) reduces Bcr-Abl-mediated vascular endothelial growth factor secretion in chronic myelogenous leukemia

A large and diverse spectrum of oncogenes has been implicated as a contributor to angiogenesis in solid tumors based, in part, on its ability to induce proangiogenic growth factors such as vascular endothelial growth factor (VEGF), and the fact that various anti-oncogenic signaling inhibitor drugs have been shown to reverse such proangiogenic effects both in vitro and in vivo. Because leukemias are now also considered to be angiogenesis-dependent malignancies, we asked whether a similar paradigm might exist for the BCR-ABL oncogene and the Bcr-Abl targeting drug, STI-571 (imatinib mesylate), in the context of chronic myelogenous leukemia (CML) cells. We found that levels of VEGF expression in BCR-ABL-positive K562 cells were reduced in vitro by treatment with STI-571 in a dose-dependent fashion. Transfection of BCR-ABL into murine myeloid 32D and human megakaryocyte MO7e hematopoietic cells resulted in enhanced VEGF expression, which could be further elevated by the exposure to cytokines such as interleukin 3 and granulocyte macrophage colony-stimulating factor. We also found that conditioned media taken from 32D-p210-transfected cells could stimulate human umbilical vein endothelial cells by increasing phosphorylation of VEGF-R2/KDR and the downstream serine/threonine kinase PKB/Akt, an important regulator of endothelial cell survival. Moreover, amplification of BCR-ABL in STI-571-resistant cells was associated with elevated VEGF expression levels which could be reversed by treatment with higher concentrations of STI-571. Taken together, our results implicate BCR-ABL as a possible regulator of CML angiogenesis and raise the possibility that STI-571 could mediate some of its anti-CML properties in vivo through an angiogenesis-dependent mechanism.     

Coordinate regulation of lymphocyte-endothelial interactions by pregnancy-associated hormones

Precursors of uterine NK cells home to the uterus during early pregnancy from multiple lymphohemopoietic sources. In mouse uterine tissue, pregnancy markedly up-regulates both L-selectin- and alpha(4) integrin-dependent adhesion pathways for circulating human CD56(bright) cells, the phenotype of human uterine NK cells. Based on roles for these adhesion molecules in lymphocyte homing, we examined effects of pregnancy or the steroid hormones 17beta-estradiol or progesterone on lymphocyte-endothelial interactions in secondary lymphoid tissues and in uterus. From preimplantation gestation day 3, specialized high endothelial venules in peripheral lymph nodes and Peyer's patches supported elevated L-selectin and alpha(4)beta(7) integrin-dependent lymphocyte adhesion under shear throughout pregnancy, as compared with high endothelial venules of virgin or postpartum donors. Squamous endothelium from nonlymphoid tissue was not affected. Pregnancy-equivalent endothelial responses were observed in lymph nodes and Peyer's patches from ovariectomized mice receiving 17beta-estradiol and/or progesterone replacement therapy. Adhesion of human CD56(bright) cells to uteri from pregnant or hormone-treated ovariectomized mice was enhanced through L-selectin- and alpha(4) integrin-dependent mechanisms and involved multiple vascular adhesion molecules including mucosal addressin cell adhesion molecule-1, VCAM-1, and peripheral lymph node addressin. Analysis of Tie2-green fluorescence protein transgenic mice demonstrated that CD56(bright) cells adhered primarily to vascular endothelium within the decidua basalis. Microdomain localization of adhesion involving large clusters of lymphocytes was induced on uteri from natural matings, but not pseudopregnancy. Steroid hormones also had independent effects on L-selectin function in splenic lymphocytes that mimicked physiological stimulation induced by pregnancy or fever-range temperatures. These results provide the first evidence for coordinated, organ-specific, steroid hormone-induced changes in lymphocyte homing mechanisms that could contribute to local and systemic immune responses during pregnancy.     

Interplay between neutrophils and trophoblast cells conditions trophoblast function and triggers vascular transformation signals

Normal placentation entails highly regulated interactions of maternal leukocytes with vascular and trophoblast cells to favor vascular transformation. Neutrophil activation and neutrophil extracellular trap (NET) formation associate with poor placentation and severe pregnancy complications. To deepen into the mechanisms of trophoblast–neutrophil interaction, we explored the effects of NETs on trophoblast cell function and, conversely, whether trophoblast cell-derived factors condition neutrophils to favor angiogenesis and anti-inflammatory signals required for fetal growth. NETs isolated from activated neutrophils hindered trophoblast cell migration. Trophoblast conditioned media prevented the effect as well as the vasoactive intestinal peptide (VIP) known to regulate trophoblast and neutrophil function. On the other hand, factors released by trophoblast cells and VIP shaped neutrophils to a proangiogenic profile with increased vascular endothelial growth factor synthesis and increased capacity to promote vascular transformation. Results presented here provide novel clues to reconstruct the interaction of trophoblast cells and neutrophils in vivo during placentation in humans.     

Adult tissue angiogenesis: evidence for negative regulation by estrogen in the uterus.

Increased uterine vascular permeability and angiogenesis are two major events of embryo implantation and placentation during pregnancy. These latter processes require coordinated, uterine-specific interactions between progesterone (P4) and estrogen (E) signaling. Although roles of these steroids have long been suspected, definitive functions of E and/or P4 in uterine angiogenesis still remain elusive. We have therefore exploited the availability of reporter and mutant mice to explore the regulation of angiogenesis in response to steroid hormonal changes in vivo. We present here molecular, genetic, physiological, and pharmacological evidence that E and P4 have different effects in vivo: E promotes uterine vascular permeability but profoundly inhibits angiogenesis, whereas P4 stimulates angiogenesis with little effect on vascular permeability. These effects of E and P4 are mediated by differential spatiotemporal expression of proangiogenic factors in the uterus.     

Hypoxia stimulates pancreatic stellate cells to induce fibrosis and angiogenesis in pancreatic cancer

Pancreatic cancer is characterized by excessive desmoplastic reaction and by a hypoxic microenvironment within the solid tumor mass. Chronic pancreatitis is also characterized by fibrosis and hypoxia. Fibroblasts in the area of fibrosis in these pathological settings are now recognized as activated pancreatic stellate cells (PSCs). Recent studies have suggested that a hypoxic environment concomitantly exists not only in pancreatic cancer cells but also in surrounding PSCs. This study aimed to clarify whether hypoxia affected the cell functions in PSCs. Human PSCs were isolated and cultured under normoxia (21% O(2)) or hypoxia (1% O(2)). We examined the effects of hypoxia and conditioned media of hypoxia-treated PSCs on cell functions in PSCs and in human umbilical vein endothelial cells. Hypoxia induced migration, type I collagen expression, and vascular endothelial growth factor (VEGF) production in PSCs. Conditioned media of hypoxia-treated PSCs induced migration of PSCs, which was inhibited by anti-VEGF antibody but not by antibody against hepatocyte growth factor. Conditioned media of hypoxia-treated PSCs induced endothelial cell proliferation, migration, and angiogenesis in vitro and in vivo. PSCs expressed several angiogenesis-regulating molecules including VEGF receptors, angiopoietin-1, and Tie-2. In conclusion, hypoxia induced profibrogenic and proangiogenic responses in PSCs. In addition to their established profibrogenic roles, PSCs might play proangiogenic roles during the development of pancreatic fibrosis, where they are subjected to hypoxia.     

Involvement of vessels and PDGFB in muscle splitting during chick limb development

Muscle formation and vascular assembly during embryonic development are usually considered separately. In this paper, we investigate the relationship between the vasculature and muscles during limb bud development. We show that endothelial cells are detected in limb regions before muscle cells and can organize themselves in space in the absence of muscles. In chick limbs, endothelial cells are detected in the future zones of muscle cleavage, delineating the cleavage pattern of muscle masses. We therefore perturbed vascular assembly in chick limbs by overexpressing VEGFA and demonstrated that ectopic blood vessels inhibit muscle formation, while promoting connective tissue. Conversely, local inhibition of vessel formation using a soluble form of VEGFR1 leads to muscle fusion. The endogenous location of endothelial cells in the future muscle cleavage zones and the inverse correlation between blood vessels and muscle suggests that vessels are involved in the muscle splitting process. We also identify the secreted factor PDGFB (expressed in endothelial cells) as a putative molecular candidate mediating the muscle-inhibiting and connective tissue-promoting functions of blood vessels. Finally, we propose that PDGFB promotes the production of extracellular matrix and attracts connective tissue cells to the future splitting site, allowing separation of the muscle masses during the splitting process.     

Endothelium-Derived 5-Methoxytryptophan Protects Endothelial Barrier Function by Blocking p38 MAPK Activation

The endothelial junction is tightly controlled to restrict the passage of blood cells and solutes. Disruption of endothelial barrier function by bacterial endotoxins, cytokines or growth factors results in inflammation and vascular damage leading to vascular diseases. We have identified 5-methoxytryptophan (5-MTP) as an anti-inflammatory factor by metabolomic analysis of conditioned medium of human fibroblasts. Here we postulated that endothelial cells release 5-MTP to protect the barrier function. Conditioned medium of human umbilical vein endothelial cells (HUVECs) prevented endothelial hyperpermeability and VE-cadherin downregulation induced by VEGF, LPS and cytokines. We analyzed the metabolomic profile of HUVEC conditioned medium and detected 5-MTP but not melatonin, serotonin or their catabolites, which was confirmed by enzyme-linked immunosorbent assay. Addition of synthetic pure 5-MTP preserved VE-cadherin and maintained barrier function despite challenge with pro-inflammatory mediators. Tryptophan hydroxylase-1, an enzyme required for 5-MTP biosynthesis, was downregulated in HUVECs by pro-inflammatory mediators and it was accompanied by reduction of 5-MTP. 5-MTP protected VE-cadherin and prevented endothelial hyperpermeability by blocking p38 MAPK activation. A chemical inhibitor of p38 MAPK, SB202190, exhibited a similar protective effect as 5-MTP. To determine whether 5-MTP prevents vascular hyperpermeability in vivo, we evaluated the effect of 5-MTP administration on LPS-induced murine microvascular permeability with Evans blue. 5-MTP significantly prevented Evans blue dye leakage. Our findings indicate that 5-MTP is a new class of endothelium-derived molecules which protects endothelial barrier function by blocking p38 MAPK.     

Effect of TIM-3 Blockade on the Immunophenotype and Cytokine Profile of Murine Uterine NK Cells

NK cells are the most abundant lymphocyte population in the feto-maternal interface during gestation. The uterine NK cells (uNK) are transient, have a unique immunophenotype and produce a number of cytokines. These cytokines play an important role in establishment and maintenance of vascular remodeling and tolerance associated with successful pregnancy. The uNK cells also express TIM-3 during gestation and blockade of TIM-3 expression results in fetal loss in mice. In this study we determined the effect of TIM-3 blockade on uNK cells. Specifically we observed surface receptor phenotype and cytokine production by uNK cells following TIM-3 blockade. Our results show that TIM-3 plays a role in regulating the uNK cells and contributes to the maintenance of tolerance at the feto-maternal interface.     

G Protein α Subunit 14 Mediates Fibroblast Growth Factor 2-Induced Cellular Responses in Human Endothelial Cells

During pregnancy, a tremendous increase in fetoplacental angiogenesis is associated with elevated blood flow. Aberrant fetoplacental vascular function may lead to pregnancy complications including pre-eclampsia. Fibroblast growth factor 2 (FGF2) and vascular endothelial growth factor A (VEGFA) are crucial regulators of fetoplacental endothelial function. G protein α subunit 14 (GNA14), a member of Gαq/11 subfamily is involved in mediating hypertensive diseases and tumor vascularization. However, little is known about roles of GNA14 in mediating the FGF2- and VEGFA-induced fetoplacental endothelial function. Using human umbilical vein endothelial cells (HUVECs) cultured under physiological chronic low oxygen (3% O₂) as a cell model, we show that transfecting cells with adenovirus carrying GNA14 complementary DNA (cDNA; Ad-GNA14) increases (p < 0.05) protein expression of GNA14. GNA14 overexpression blocks (p < 0.05) FGF2-stimulated endothelial migration, whereas it enhances (p < 0.05) endothelial monolayer integrity (maximum increase of ~35% over the control at 24 hr) in response to FGF2. In contrast, GNA14 overexpression does not significantly alter VEGFA-stimulated cell migration, VEGFA-weakened cell monolayer integrity, and intracellular Ca⁺⁺ mobilization in response to adenosine triphosphate (ATP), FGF2, and VEGFA. GNA14 overexpression does not alter either FGF2- or VEGFA-induced phosphorylation of ERK1/2. However, GNA14 overexpression time-dependently elevates (p < 0.05) phosphorylation of phospholipase C-β3 (PLCβ3) at S1105 in response to FGF2, but not VEGFA. These data suggest that GNA14 distinctively mediates fetoplacental endothelial cell migration and permeability in response to FGF2 and VEGFA, possibly in part by altering activation of PLCβ3 under physiological chronic low oxygen.     

Adrenomedullin stimulates angiogenic response in cultured human vascular endothelial cells: involvement of the vascular endothelial growth factor receptor 2

In recent years, evidence has accumulated that many endogenous peptides play an important regulatory role in angiogenesis by modulating endothelial cell behavior. Adrenomedullin (AM), one such factor, was previously shown to exert a clearcut proangiogenic effect in vitro when tested on specialized human endothelial cells, such as HUVECs and immortalized endothelial cell lines. In the present study we used normal adult vascular endothelial cells isolated from human saphenous vein to analyze in vitro the role of AM, related to both early (increased cell proliferation) and late (differentiation and self-organization into capillary-like structures) angiogenic events and their relationship with the vascular endothelial growth factor (VEGF) signaling cascade. The results indicated that also in this endothelial cell phenotype AM promoted cell proliferation and differentiation into cord-like structures. These actions resulted specific and were mediated by the binding of AM to its AM1 (CRLR/RAMP2) receptor. Neither the administration of a VEGF receptor 2 (VEGFR-2) antagonist nor the downregulation of VEGF production by gene silencing were able to suppress the proangiogenic effect of AM. However, when the experiments were performed in the presence of SU5416 (a selective inhibitor of the VEGFR-2 receptor at the level of the intra-cellular tyrosine kinase domain) the proangiogenic effect of AM was abolished. This result suggests that in vascular endothelial cells the binding of AM to its AM1 receptor could trigger a transactivation of the VEGFR-2 receptor, leading to a signaling cascade inducing proangiogenic events in the cells.